The clinical use of high dose methotrexate in conjunction with citrovorum factor "rescue", especially in treatment of solid tumors, has necessitated a re-evaluation of the pharmacokinetics of methotrexate, particularly with regard to the metabolism of this agent in humans. We have undertaken studies aimed at characterizing the enzyme in human liver responsible for oxidation of methotrexate to 7-hydroxymethotrexate; our investigations involve development and application of high sensitive (e.g. isotopic and fluorometric) analytical techniques for detection and quantitation of this reaction. In addition, we have had a long standing interest in lipid-soluble antifolates, which differ from methotrexate both in cellular transport properties and in susceptibility to enzymatic oxidation. In anticipation of possible pharmacokinetic studies with a series of new antifolates (2,4-diaminopyrroloquinazolines) we have developed sensitive assays (fluorescence and enzyme inhibition) for these compounds.